"The ability of cells to invade underlies many pathological conditions such as metastasis and inflammation. Invasion requires coordination of changes in the biology of the invading cell coupled with changes in the adhesive behavior and integrity of the penetrated barrier. This proposed project aims to understand the modulation of epithelial integrity during immune cell transmigration. Recent work has shown that Drosophila hemocytes invade through an epithelial barrier during embryonic development and require the small GTPase RhoL to breach Cadherin barriers. We will identify morphological and biophysical changes in epithelial integrity during this immune cell transmigration. To assess potential changes in junctional Cadherin expression during hemocyte invasion, immunofluorescence analysis and live imaging of junctional DE-Cadherin and the Actomyosin network will be performed. This will be followed by “Fluorescence Recovery after Photobleaching” (FRAP) studies of DE-Cadherin-GFP fusion proteins to understand whether hemocyte invasion alters DE-Cadherin stability at the junctions. These experiments will provide clear insight into whether changes in DE-Cadherin integrity precede hemocyte invasion. Cell-cell junctions are known sites of mechanotransduction and hence this project will next aim to evaluate possible changes in the mechanobiology of Adherens junctions during transmigration. A FRET based tension sensor will be cloned into Drosophila and utilized to assess if there is force generation at the Adherens junctions during transmigration. This will be followed by laser nanoablation of cell junctions to assess changes in cortical tension during hemocyte invasion. These studies will concertedly address the role of junctional changes and mechanotransduction during invasion and thus provide new insight into epithelial biology during immune cell transmigration. Our studies should have relevance for future studies of pathologies like inflammation and cancer metastasis."